A magnesium alendronate nano-preparation and its application in metabolic-related fatty liver disease

Abstract

This invention belongs to the field of biomedical technology, and specifically relates to an alendronate magnesium nanoparticle formulation and its application in metabolic-associated fatty liver disease (MAFLD). The alendronate magnesium (MgALN) nanoparticle formulation of this invention is formed by the coordination of alendronate sodium and magnesium ions. The prepared MgALN nanoparticle formulation has an average particle size of approximately 452 nm, exhibiting good dispersibility and stability, achieving highly efficient targeted enrichment of the drug in the liver, and significantly improving the drug's retention time and local concentration in the liver. This formulation effectively inhibits lipid accumulation in hepatocytes, improves the pathological phenotype of fatty liver in mice induced by a high-fat diet, and has no significant cytotoxicity or systemic toxicity in vivo. This invention solves the problems of insufficient liver targeting, low bioavailability, and limited efficacy of traditional alendronate sodium in treating MAFLD, providing a novel and highly effective targeted drug for the clinical treatment of MAFLD, and has significant clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an alendronate magnesium nanoparticle formulation and its application in metabolism-related fatty liver disease. Background Technology

[0002] Metabolic fatty liver disease (MAFLD) is a globally prevalent chronic liver disease that represents the liver's manifestation of systemic metabolic dysfunction. Its spectrum includes simple fatty liver, metabolic dysfunction-associated steatohepatitis (MASH), liver fibrosis, cirrhosis, and liver cancer. In recent years, the prevalence of MAFLD in China has been steadily increasing, becoming the leading cause of chronic liver disease in the country, seriously endangering public health and imposing a heavy socioeconomic burden.

[0003] Currently, the main prevention and treatment measures for MAFLD are lifestyle interventions, but poor patient adherence leads to unsatisfactory results. Therefore, the development of targeted therapies has significant clinical value. Due to the complex pathogenesis and progression mechanisms of MAFLD, the identification of therapeutic targets and progress in drug development have been hampered. Apart from resimeltiro, approved by the FDA for MASH patients, no other new drugs targeting MAFLD have been approved globally. Finding new therapeutic targets and developing novel targeted drugs are crucial for the precision treatment of MAFLD.

[0004] Alendronate sodium (NaALN) is a widely used bisphosphonate drug in clinical practice, with good biocompatibility and well-defined pharmacokinetic characteristics. However, traditional NaALN primarily targets bone tissue in vivo, with limited accumulation in the liver, making it difficult to effectively intervene in hepatic lipid metabolism. Furthermore, its salt form is rapidly cleared from the bloodstream, resulting in low bioavailability. Due to the lack of precise hepatic delivery, increasing the dosage can increase potential gastrointestinal or renal side effects, severely limiting its application in the treatment of MAFLD.

[0005] Therefore, while maintaining the good safety profile of alendronate, developing an alendronate derivative formulation with strong liver targeting, high bioavailability, and significant therapeutic effect on MAFLD has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, such as insufficient liver targeting of sodium alendronate, low bioavailability, limited efficacy in treating MAFLD, and the potential for side effects with increased dosage, this invention aims to provide a method for preparing magnesium alendronate nanoparticles and their application in the treatment of metabolic-related fatty liver disease. Through metal coordination and nanotechnology, MgALN nanoparticles with good dispersibility and stability are prepared, achieving highly efficient drug targeting to the liver and providing a novel and highly effective targeted drug for the clinical treatment of MAFLD.

[0007] To achieve the above objectives, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides the application of alendronate magnesium nanoparticles in the preparation of drugs for treating metabolic-related fatty liver disease, wherein the alendronate magnesium nanoparticles are prepared by forming a nanostructure from alendronate sodium and magnesium ions through coordination.

[0008] Preferably, the preparation method of the magnesium alendronate nanoparticle formulation includes the following steps: Sodium alendronate trihydrate and magnesium sulfate were dissolved in water, and the pH was adjusted to 5-7. The mixture was stirred continuously in a water bath at 2-8°C for 25-35 minutes. After the reaction was completed, the product was collected by centrifugation and washed with anhydrous ethanol to remove impurities, thus obtaining the magnesium alendronate nano-formulation.

[0009] Preferably, the mass ratio of sodium alendronate trihydrate to magnesium sulfate is (2-4):1.

[0010] Preferably, 0.03-0.07M NaOH is used for pH adjustment.

[0011] Preferably, the effective concentration of the magnesium alendronate nanoparticle formulation is 12.5-100 mg / L.

[0012] More preferably, the effective concentration of the magnesium alendronate nanoformulation is 12.5 mg / L.

[0013] Preferably, the magnesium alendronate nanoparticle formulation can target and accumulate in liver tissue, prolonging the circulation time of the drug in the body and increasing the local concentration of the drug in the liver.

[0014] The MgALN nanoparticle formulation prepared by the above method has a strong liver-targeting ability. After being injected via the tail vein, it has a long circulation time in the body and can preferentially accumulate in liver tissue, significantly increasing the local drug concentration in the liver and providing sufficient drug concentration for the treatment of MAFLD. At the same time, the formulation can effectively inhibit lipid accumulation in hepatocytes, improve hepatic steatosis, reduce body weight, liver weight and liver-to-body ratio in fatty liver mice, correct dyslipidemia, and bring serum ALT, AST and other liver function indicators back to normal levels, significantly improving the pathological phenotype of MAFLD.

[0015] In addition, MgALN nanoparticles are derived from clinically used NaALN and inherit its good biocompatibility: they have no obvious toxic side effects on AML-12 hepatocytes in the concentration range of 12.5-100 mg / L, and cell viability remains at a high level; at the in vivo dosage, they do not cause obvious tissue structural abnormalities or inflammatory reactions in major organs such as the heart, lungs, spleen, and kidneys, and have no systemic toxicity, making them highly safe to use.

[0016] Furthermore, when preparing a drug for treating MAFLD using this MgALN nanoformulation, it is preferable to prepare it as an injectable formulation, with a recommended dosage of 2 mg / kg / q4d. This dosage can achieve significant therapeutic effects without the need for high-dose administration, thus avoiding the side effects caused by high doses of traditional NaALN.

[0017] Secondly, the present invention provides a method for preparing magnesium alendronate nanoparticles, comprising the following steps: Sodium alendronate trihydrate and magnesium sulfate were dissolved in water, and the pH was adjusted to 5-7. The mixture was stirred continuously in a water bath at 2-8°C for 25-35 minutes. After the reaction was completed, the product was collected by centrifugation and washed with anhydrous ethanol to remove impurities, thus obtaining the magnesium alendronate nano-formulation.

[0018] More preferably, the specific reaction conditions are: continuous stirring in a 4°C water bath for 30 min.

[0019] Preferably, the mass ratio of sodium alendronate trihydrate to magnesium sulfate is (2-4):1.

[0020] More preferably, the mass ratio of sodium alendronate trihydrate to magnesium sulfate is 3:1.

[0021] Preferably, 0.03-0.07M NaOH is used for pH adjustment.

[0022] The preferred preparation process parameters and raw material ratios described in this invention play a crucial role in obtaining alendronate magnesium nanoparticles with excellent dispersibility, stable structure, and liver-targeting potential. By controlling the reaction pH to 5-7, a low-temperature (2-8℃) water bath, and a specific stirring time, the coordination assembly process of sodium alendronate and magnesium ions can be precisely regulated, avoiding nanoparticle aggregation and significantly improving the uniformity and dispersibility of the formulation. The preferred mass ratio of (2-4):1 and purification by washing with anhydrous ethanol can effectively remove unreacted impurities, enhance the crystallinity and storage stability of the nanostructure, and reduce in vivo degradation and premature release. At the same time, the regular and controllable nanomorphology and magnesium-based carrier characteristics can improve the retention capacity of the formulation in the blood circulation, enhance liver tissue enrichment and targeted delivery efficiency, thereby providing a stable and efficient drug delivery basis for the precise treatment of metabolic-related fatty liver disease, ensuring subsequent efficacy and clinical translation feasibility.

[0023] Thirdly, the present invention provides alendronate magnesium nanoparticles prepared by the aforementioned preparation method.

[0024] The nano-formulation described in this invention is formed by coordination between sodium alendronate and magnesium ions to create a nanostructure. The resulting MgALN nano-formulation has an average particle size of approximately 452 nm, exhibits a unimodal particle size distribution, and demonstrates good dispersibility. Its zeta potential is -5.5 mV, and its negatively charged surface facilitates stable dispersion in aqueous systems and prevents aggregation. This nano-formulation significantly enhances enrichment and retention in liver tissue while possessing good biocompatibility, providing a reliable basis for targeted drug delivery to the liver.

[0025] Fourthly, the present invention provides a medicament for treating metabolic-related fatty liver disease, the medicament comprising the aforementioned magnesium alendronate nanoformulation, and pharmaceutically acceptable excipients.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Strong liver targeting and high bioavailability: This invention constructs a nanostructure through the coordination of magnesium ions and sodium alendronate, which changes the spatial conformation and physicochemical properties of the original molecule, so that the MgALN nano-formulation gets rid of the bone targeting characteristics of traditional NaALN and achieves efficient targeted enrichment of the liver; the formulation has a long circulation time in vivo and significantly improves the liver retention capacity, greatly increasing the local concentration of the drug in the liver, and solving the technical problems of insufficient liver targeting and low bioavailability of NaALN.

[0027] (2) Significant therapeutic effect and clear mechanism of action: MgALN nanoparticles can effectively inhibit lipid accumulation in hepatocytes, significantly improve the pathological phenotype of fatty liver in mice induced by high-fat diet, reduce mouse body weight, liver weight and liver-to-body ratio, correct blood lipid disorders, restore liver function indicators, reduce hepatic steatosis and lipid deposition, and have a better therapeutic effect on MAFLD than traditional NaALN, and the dosage is lower, so effective treatment can be achieved without high doses.

[0028] (3) Good biosafety and no obvious toxic side effects: MgALN nano-formulation is derived from NaALN, which is widely used in clinical practice, and inherits its good biosafety. In vitro experiments show that it has no obvious toxicity to hepatocytes within the effective dosage range, and in vivo experiments show that it has no obvious damage to major organs such as heart, lungs, spleen, and kidneys, and has no systemic toxicity. This solves the problem that traditional NaALN is prone to gastrointestinal and renal side effects when the dosage is increased.

[0029] (4) The preparation method is simple and has good repeatability: The preparation process of the MgALN nano-formulation of the present invention only requires four steps: raw material dissolution, mixing and pH adjustment, low temperature reaction, separation and purification. The operation is simple, the reaction conditions are mild, no complicated instruments and equipment are required, the product has high purity, uniform particle size distribution, good repeatability, and is suitable for large-scale production. It has good industrial application prospects. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the preparation process of the magnesium alendronate nanoformulation of the present invention; Figure 2 This is a particle size distribution diagram of the MgALN nanoparticles of the present invention; Figure 3 The results of the effect of NaALN / MgALN on the viability of AML-12 cells are shown in Figure A, where different concentrations of NaALN and their combination with Model have an effect on the viability of AML-12 cells, and different concentrations of MgALN and their combination with Model have an effect on the viability of AML-12 cells. Figure 4 Oil Red staining image showing the inhibitory effect of NaALN / MgALN on lipid accumulation in AML-12 cells (white light image, scale bar in the lower right corner is 100 μm). Figure 5 This is a quantitative analysis of relative lipid content in AML-12 cells after Oil Red staining. The calculation formula is as follows: Relative lipid content = (OD490 of the drug-treated group - OD490 of the normal group) / (OD490 of the model group - OD490 of the normal group) x 100%. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Figure 6 This diagram shows the fluorescence tracing and tissue distribution analysis of MgALN in mice according to the present invention. A represents in vivo fluorescence imaging of mice in the free DiR group and the DiR-MgALN group at 0, 3, 6, 24, 48, and 72 hours after drug administration; B represents in vitro fluorescence imaging of major organs of mice after drug administration; C represents the quantitative results of fluorescence signal changes over time in the free DiR group; D represents the quantitative results of fluorescence signal changes over time in the DiR-MgALN group; E represents the quantitative analysis results of fluorescence signal intensity in each organ; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Figure 7 This diagram illustrates the therapeutic effect of MgALN on a high-fat diet-induced fatty liver mouse model. A shows the establishment of the MAFLD model and drug administration; B shows the mouse body weight change curve; C shows the area under the body weight change curve; D shows the mouse liver as seen from a macroscopic perspective; E shows the mouse liver weight; F shows the mouse liver-to-body ratio; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Figure 8 The images show the pathological changes and safety evaluation of MgALN in the liver tissue of mice with fatty liver, where A is the H&E staining image of mouse liver tissue; B is the Oil Red O staining image of mouse liver tissue; and C is the H&E staining image of major organs of mice. Detailed Implementation

[0031] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0032] Example 1 This embodiment provides a method for preparing magnesium alendronate nanoparticles, including the following steps: (1) Take 0.3g of sodium alendronate trihydrate, add 40mL of deionized water, and stir thoroughly until completely dissolved to obtain sodium alendronate aqueous solution; take 0.1g of magnesium sulfate, add 40mL of deionized water, and stir thoroughly until completely dissolved to obtain magnesium sulfate aqueous solution; (2) Pour the above sodium alendronate and magnesium sulfate aqueous solution into the same reaction vessel, mix them evenly under magnetic stirring, and then slowly add 0.05M NaOH solution, monitor the pH value of the mixed system in real time until the pH value is adjusted to 6.0; (3) Place the pH-adjusted reaction solution in a 4°C water bath and keep it magnetically stirred for 30 minutes to allow magnesium ions to fully coordinate with sodium alendronate. (4) After the reaction is complete, the reaction solution is transferred to a centrifuge tube and centrifuged at 8000 r / min for 10 min. The bottom precipitate is collected. Anhydrous ethanol is added to the precipitate, and after thorough shaking, it is centrifuged again. The supernatant is discarded, and the washing is repeated 4 times to remove unreacted raw materials and impurities. Finally, magnesium alendronate (MgALN) nanoparticles are obtained.

[0033] Example 2 This embodiment provides a method for preparing magnesium alendronate nanoparticles, including the following steps: (1) Take 0.2g of alendronate sodium trihydrate, add 40mL of deionized water, and stir thoroughly until completely dissolved to obtain an aqueous solution of alendronate sodium; take another 0.1g of magnesium sulfate, add 40mL of deionized water, and stir thoroughly until completely dissolved to obtain an aqueous solution of magnesium sulfate; (2) Pour the above sodium alendronate and magnesium sulfate aqueous solution into the same reaction vessel, mix them evenly under magnetic stirring, and then slowly add 0.05M NaOH solution, monitoring the pH value of the mixed system in real time until the pH value is adjusted to 5.0; (3) Place the pH-adjusted reaction solution in a 2°C water bath and keep it magnetically stirred for 35 minutes to allow magnesium ions to fully coordinate with sodium alendronate. (4) After the reaction is complete, the reaction solution is transferred to a centrifuge tube and centrifuged at 8000 r / min for 10 min. The bottom precipitate is collected. Anhydrous ethanol is added to the precipitate, and after thorough shaking, it is centrifuged again. The supernatant is discarded, and the washing is repeated 4 times to remove unreacted raw materials and impurities. Finally, magnesium alendronate (MgALN) nanoparticles are obtained.

[0034] Example 3 This embodiment provides a method for preparing magnesium alendronate nanoparticles, including the following steps: (1) Take 0.4g of sodium alendronate trihydrate, add 40mL of deionized water, and stir thoroughly until completely dissolved to obtain sodium alendronate aqueous solution; take another 0.1g of magnesium sulfate, add 40mL of deionized water, and stir thoroughly until completely dissolved to obtain magnesium sulfate aqueous solution; (2) Pour the above sodium alendronate and magnesium sulfate aqueous solution into the same reaction vessel, mix them evenly under magnetic stirring, and then slowly add 0.05M NaOH solution, monitoring the pH value of the mixed system in real time until the pH value is adjusted to 7.0; (3) Place the pH-adjusted reaction solution in an 8°C water bath and keep it magnetically stirred for 25 minutes to allow magnesium ions to fully coordinate with sodium alendronate. (4) After the reaction is complete, the reaction solution is transferred to a centrifuge tube and centrifuged at 8000 r / min for 10 min. The bottom precipitate is collected. Anhydrous ethanol is added to the precipitate, and after thorough shaking, it is centrifuged again. The supernatant is discarded, and the washing is repeated 4 times to remove unreacted raw materials and impurities. Finally, magnesium alendronate (MgALN) nanoparticles are obtained.

[0035] Comparative Example 1 This comparative example provides a method for preparing magnesium alendronate nanoparticles, including the following steps: (1) Take 0.1g of sodium alendronate trihydrate, add 40mL of deionized water, and stir thoroughly until completely dissolved to obtain sodium alendronate aqueous solution; take another 0.1g of magnesium sulfate, add 40mL of deionized water, and stir thoroughly until completely dissolved to obtain magnesium sulfate aqueous solution; (2) Pour the above sodium alendronate and magnesium sulfate aqueous solutions into the same reaction vessel, mix them evenly under magnetic stirring, and then slowly add 0.05M NaOH solution, monitoring the pH value of the mixture in real time until the pH value is adjusted to 8.0; (3) Place the pH-adjusted reaction solution in a 10°C water bath and keep it magnetically stirred for 15 minutes. (4) After the reaction is complete, the reaction solution is transferred to a centrifuge tube and centrifuged at 8000 r / min for 10 min. The bottom precipitate is collected. Anhydrous ethanol is added to the precipitate, and after thorough shaking, it is centrifuged again. The supernatant is discarded, and the washing is repeated 4 times to remove unreacted raw materials and impurities. Finally, magnesium alendronate (MgALN) nanoparticles are obtained.

[0036] Comparative Example 2 This comparative example provides a method for preparing magnesium alendronate nanoparticles, including the following steps: (1) Take 0.5g of sodium alendronate trihydrate, add 40mL of deionized water, and stir thoroughly until completely dissolved to obtain sodium alendronate aqueous solution; take another 0.1g of magnesium sulfate, add 40mL of deionized water, and stir thoroughly until completely dissolved to obtain magnesium sulfate aqueous solution; (2) Pour the above sodium alendronate and magnesium sulfate aqueous solution into the same reaction vessel, mix them evenly under magnetic stirring, and then slowly add 0.05M NaOH solution, monitoring the pH value of the mixed system in real time until the pH value is adjusted to 5.0; (3) Place the pH-adjusted reaction solution in a 10°C water bath and keep it magnetically stirred for 40 min. (4) After the reaction is complete, the reaction solution is transferred to a centrifuge tube and centrifuged at 8000 r / min for 10 min. The bottom precipitate is collected. Anhydrous ethanol is added to the precipitate, and after thorough shaking, it is centrifuged again. The supernatant is discarded, and the washing is repeated 4 times to remove unreacted raw materials and impurities. Finally, magnesium alendronate (MgALN) nanoparticles are obtained.

[0037] Example of effect 1 This example uses MgALN nanoparticles prepared by the methods in Example 1 and Comparative Examples 1-2 as test samples to characterize their particle size and physicochemical properties of Zeta potential. The specific methods are as follows: The MgALN nanoparticles prepared in Example 1 and Comparative Examples 1-2 were dispersed in deionized water and ultrasonically dispersed for 5 min to obtain a uniform suspension. An appropriate amount of sample was placed in a quartz cuvette, and the particle size distribution was measured at 25°C using a dynamic light scattering (DLS) instrument. The Zeta potential was measured using electrophoretic light scattering. Each sample was measured three times, and the average value was taken.

[0038] The results showed that the average particle size of the MgALN nanoparticles described in Example 1 was about 452 nm, the particle size distribution curve showed a single peak, and the dispersion was good. The zeta potential was -5.5 mV, the surface was negatively charged, and the nanoparticles had good stability in the aqueous system and were not easy to agglomerate. Figure 2The image shows the particle size distribution of MgALN as described in Example 1. The average particle size of the MgALN nanoparticles in Comparative Example 1 is approximately 734 nm, with a Zeta potential of -2.5 mV; the average particle size of the MgALN nanoparticles in Comparative Example 2 is approximately 674 nm, with a Zeta potential of -2.8 mV. These results indicate that when the preparation process exceeds the preferred range of this invention, the resulting MgALN nanoparticles exhibit a significantly increased particle size, decreased particle size uniformity, and a markedly reduced absolute value of the Zeta potential. The insufficient surface negative charge density leads to a weakened electrostatic repulsion of the nanoparticles in the aqueous phase, decreased stability, and a greater susceptibility to aggregation and sedimentation.

[0039] By controlling the preparation process within a preferred range, this invention can precisely regulate the particle size, particle size distribution, and surface potential of MgALN nanoparticles, resulting in nano-formulations with smaller particle size, better uniformity, and superior dispersibility and aqueous phase stability. This lays a key formulation foundation for better efficacy, improved bioavailability, and therapeutic effects on metabolic-related fatty liver disease in vivo.

[0040] Example 2 This example uses the MgALN nanoparticles prepared in Example 1 as the test sample to detect their cellular biocompatibility. The specific method is as follows: AML-12 hepatocytes were cultured in DMEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. After cell adhesion and stability, they were seeded into 96-well plates, and different concentrations of MgALN (12.5, 25, 50, and 100 mg / L) were added, with a blank control group included. An AML-12 cell model of lipid accumulation was established by inducing OA / PA (2:1) treatment, with the same concentrations of MgALN added. A control group with the same concentration gradient of NaALN was also included to compare the cytotoxicity of the two drugs. After 24 h of treatment, cell viability was assessed using the CCK-8 assay. Working solution was added according to the kit instructions, and incubation continued for 30 minutes. Absorbance was then measured at 450 nm using a microplate reader. Relative cell viability was calculated as the ratio of OD values ​​of the experimental and control groups. Each experiment was independently repeated five times (n=5), and results are expressed as mean ± standard deviation.

[0041] Experimental results showed that within the dosage range of 12.5-100 mg / L, cell viability remained at a high level in all MgALN-treated groups, with no significant difference compared to the blank control group (P>0.05). This indicates that the MgALN nanoparticle formulation has no obvious toxic side effects on hepatocytes and exhibits good biosafety (see...). Figure 3 ).

[0042] Example 3 This example uses the MgALN nanoparticles prepared in Example 1 as the test sample to detect their lipid metabolism regulation ability. The specific method is as follows: A lipid accumulation cell model was established by inducing AML-12 cells with OA / PA (2:1). The model cells were divided into a Model group, a NaALN treatment group (12.5, 25, 50, 100 mg / L), and a MgALN treatment group (12.5, 25, 50, 100 mg / L). A normal hepatocyte control group was also set up. The corresponding concentration of drug was added to each treatment group. After culturing for 24 h, the intracellular lipid accumulation was detected by Oil Red O staining kit, and the cells were observed and photographed under a microscope. Then, the lipid droplets were dissolved in 100% isopropanol, and the absorbance was measured at a wavelength of 490 nm to calculate the relative lipid content.

[0043] The experimental results showed that a large number of red lipid droplets appeared in the hepatocytes of the Model group, indicating severe lipid accumulation. The number and size of lipid droplets decreased in the NaALN-treated group, while the reduction was more significant in the MgALN-treated group. The 12.5 mg / L MgALN-treated group had the lowest relative lipid content, showing a highly significant difference compared to the Model group (P < 0.0001). This indicates that MgALN nanoparticles can effectively inhibit lipid accumulation in hepatocytes, improve lipid metabolism disorders, and have a superior lipid metabolism regulation ability compared to traditional NaALN (see...). Figure 4-5 ).

[0044] Example of effect 4 This example uses the MgALN nanoparticles prepared in Example 1 as the test sample to study their in vivo fluorescence tracing and tissue distribution. The specific methods are as follows: To investigate the distribution behavior of MgALN nanoparticles in vivo and their liver enrichment characteristics, MgALN nanoparticles were labeled using the near-infrared fluorescent probe DiR, and in vivo tracking and ex vivo organ distribution analysis were performed using a small animal in vivo fluorescence imaging system.

[0045] Near-infrared fluorescent probe DiR was dissolved in an organic solvent, and a dispersion of MgALN nanoparticles was added. The mixture was incubated under light-protected conditions to load DiR onto the surface of the MgALN nanoparticles. Unbound free DiR was then removed by centrifugation and washing to obtain DiR-labeled MgALN nanoparticles (DiR-MgALN).

[0046] Healthy KM mice (weighing 18-22g) were randomly divided into a free DiR group and a DiR-MgALN group. Each group received the drug via tail vein injection at a dose of 5 mg / kg. In vivo fluorescence imaging was performed at 0h, 3h, 6h, 24h, 48h, and 72h post-administration. Mice were anesthetized with isoflurane before imaging, and fluorescence signals were collected under the same excitation and emission parameters.

[0047] Finally, the mice were sacrificed at the end of the experiment, and the major organs, including the heart, liver, spleen, lungs, kidneys, and bone tissue, were isolated for in vitro fluorescence imaging. The fluorescence signal intensity of each organ region was then quantitatively analyzed using imaging system software.

[0048] The experimental results showed that in the free DiR group, the fluorescence signal in mice reached a high level 3 hours after drug administration, then gradually decreased over time, and weakened significantly at 72 hours, indicating that the free fluorescent probe had a short residence time in vivo. In contrast, in the DiR-MgALN group, the fluorescence signal in mice rapidly increased 3 hours after drug administration and remained at a high level from 6 to 48 hours, maintaining a stable signal until 72 hours, indicating that MgALN nanoparticles have good stability and a long circulation time in vivo.

[0049] In vitro organ fluorescence imaging results showed that, compared with the free DiR group, the DiR-MgALN group exhibited significantly enhanced fluorescence signals in the liver, and also showed relatively obvious signals in the spleen and bone tissue. Quantitative analysis of the fluorescence signals in each organ showed that the fluorescence intensity of the DiR-MgALN group in the liver was significantly higher than that in the free DiR group (P < 0.0001), and also significantly increased in bone tissue (P < 0.0001) (see...). Figure 6 The above results indicate that MgALN nanoparticles exhibit significant liver enrichment characteristics in vivo. The signal in the spleen may be related to the uptake of nanoparticles by the reticuloendothelial system, while the fluorescence signal in bone tissue may be related to the high affinity of MgALN derived from NaALN for hydroxyapatite in bone tissue.

[0050] This demonstrates that MgALN nanoparticles can achieve stable circulation in vivo and preferentially accumulate in liver tissue, thereby improving the local concentration of the drug in the liver and providing experimental evidence for its application in the treatment of MAFLD.

[0051] Example 5 This effect example uses MgALN nanoparticles prepared in Example 1 as the test sample to explore their therapeutic effect on a mouse model of fatty liver. The specific methods are as follows: Eight-week-old C57BL / 6 mice were randomly divided into four groups: normal diet group (ND group), high-fat diet group (HFD group), high-fat diet + NaALN group (HFD+NaALN group), and high-fat diet + MgALN group (HFD+MgALN group). Except for the ND group, the other groups were given a high-fat diet to induce a fatty liver model. Four months after the model was established, drug intervention was carried out. The HFD+NaALN group was given 20 mg / kg / q4d, and the HFD+MgALN group was given 2 mg / kg / q4d. The drug administration lasted for 2 months, and the weight of the mice was monitored regularly during the period.

[0052] Mice were sacrificed at the end of the experiment, and liver weight and liver-to-body ratio were measured. Serum was collected to detect liver function indicators such as ALT and AST. Liver tissue was separated for H&E staining and Oil Red O staining to observe liver pathological changes and lipid deposition. At the same time, the heart, lungs, spleen, and kidneys were separated for H&E staining to evaluate the in vivo toxicity of the drug.

[0053] Experimental results showed that, compared with the ND group, mice in the HFD group had significantly increased body weight, enlarged and lighter-colored livers, significantly increased liver weight and liver-to-body ratio, significantly increased serum ALT and AST levels, and obvious fatty degeneration, inflammatory infiltration, and large amounts of lipid deposition in the liver tissue, indicating a successful establishment of the fatty liver model. Compared with the HFD group, mice in the HFD+MgALN group showed a significantly reduced trend in body weight gain, significantly decreased liver weight and liver-to-body ratio, returned serum ALT and AST levels to normal, significantly reduced fatty degeneration and lipid deposition in the liver tissue, and significantly improved pathological structure, demonstrating a superior therapeutic effect compared to the HFD+NaALN group. Furthermore, no obvious tissue structural abnormalities or inflammatory responses were observed in the heart, lungs, spleen, and kidneys of mice in any group, indicating that the MgALN nanoparticle formulation has a significant therapeutic effect on MAFLD and good in vivo biosafety (see [link to relevant documentation]). Figure 7-8 ).

[0054] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of an alendronate magnesium nanoparticle formulation in the preparation of a drug for treating metabolic-related fatty liver disease, characterized in that, The magnesium alendronate nanoformulation is prepared by forming a nanostructure from sodium alendronate and magnesium ions through coordination.

2. The application as described in claim 1, characterized in that, The preparation method of the magnesium alendronate nanoparticle formulation includes the following steps: Sodium alendronate trihydrate and magnesium sulfate were dissolved in water, and the pH was adjusted to 5-7. The mixture was stirred continuously in a water bath at 2-8°C for 25-35 minutes. After the reaction was completed, the product was collected by centrifugation and washed with anhydrous ethanol to remove impurities, thus obtaining the magnesium alendronate nano-formulation.

3. The application as described in claim 2, characterized in that, The mass ratio of sodium alendronate trihydrate to magnesium sulfate is (2-4):

1.

4. The application as described in claim 2, characterized in that, pH was adjusted using 0.03-0.07M NaOH.

5. The application as described in claim 1, characterized in that, The effective concentration of the magnesium alendronate nanoparticle formulation is 12.5-100 mg / L.

6. The application as described in claim 1, characterized in that, The alendronate magnesium nanoparticle formulation is targeted and enriched in liver tissue, prolonging the circulation time of the drug in the body and increasing the local concentration of the drug in the liver.

7. A method for preparing magnesium alendronate nanoparticles, characterized in that, Includes the following steps: Sodium alendronate trihydrate and magnesium sulfate were dissolved in water, and the pH was adjusted to 5-7. The mixture was stirred continuously in a water bath at 2-8°C for 25-35 minutes. After the reaction was completed, the product was collected by centrifugation and washed with anhydrous ethanol to remove impurities, thus obtaining the magnesium alendronate nano-formulation.

8. The preparation method according to claim 7, characterized in that, The mass ratio of sodium alendronate trihydrate to magnesium sulfate is (2-4):

1.

9. Magnesium alendronate nanoformulation prepared by the preparation method as described in claim 7 or 8.

10. A drug for treating metabolic-related fatty liver disease, characterized in that, The drug comprises the magnesium alendronate nanoformulation of claim 9.

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